Zone electrophoresis in capillaries has become an important technique in the repertoire of liquid-phase separations. See Jorgenson et al., Science 222 (1983) 266-272; Gordon et al., Science 242 (1988), 224-228; Ewing et al., Anal Chem. 61 (1989), 292A-303A; Wallingford et al., Advances in Chromatography 29 (1989), 1-76; and Kuhr, Anal. Chem., 62 (1990), 403R-414R. Capillary electrophoresis has been used for separations of small and large molecules and comprises several subtechniques including capillary zone electrophoresis (CZE), capillary gel electrophoresis, micellar electrokinetic capillary electrophoresis, and capillary isoelectric focusing. CZE employs extremely high potential fields, typically 300 V/cm, resulting in highly efficient separations of ionic solutes.
A major aspect of CZE in need of new development is detection; specifically, there is a critical need for detectors capable of responding to the small quantity of sample component in the effective detection volume. Detection schemes developed to date include direct and indirect UV absorption (Hjerten, J. Chromatogr., 347 (1985) 191-198 and Hjerten et al. J. Chromatogr., 403 (1987), 47-61), fluorescence (Jorgenson et al., Anal. Chem., 53 (1981), 1298-1302 and Kuhr et al., Anal. Chem., 60 (1988), 2642-2644)), and radioisotope (Pentoney et al., Anal. Chem., 61 (1989), 1642-1647) as well as mass spectrometric (Smith et al., Anal. Chem., 60 (1988), 436-441; Lee et al., Biomed. Environ. Mass Spectrom., 18 (1989), 844-850; Moseley et al., Chromatogr., 480 (1989), 197-210; Caprioli et al., J. Chromatogr., 480 (1989), 247-258)), and electrometric (Mikkers et al., J. Chromatogr., 169 (1979), 11- 20; Huang et al., Anal. Chem., 59 (1987), 2747-2749; and Wallingford et al., Anal. Chem., 59 (1987), 1762-1766)) detectors.
Electrical detectors can be distinguished from electrochemical detectors. Electrochemical detectors involve electric effects due to chemical changes which occur when a particle or species enters the detection zone. Electrical detectors respond to changes in the conductance of current or changes of resistance which result when particles or species enter the detection zone. With electrical detectors no chemical reaction is necessarily associated or required. Existing electrical and electrochemical detectors for CZE use elaborate on-column and post-column detection schemes to prevent the high separation potentials used from interfering with the detection process. One scheme involves construction of 40-.mu.m-diameter holes in the capillary using a laser. Thereafter, small platinum wire electrodes are placed in these holes to carry out on-column conductivity detection. It was demonstrated that the exact placement of these electrodes on opposite sides of the capillary is critical to minimize background noise associated with the high potential field used for separation. (Huang et al., Anal. Chem., 59 (1987), 2747-2749). In U.S. patent. application Ser. No. 443,059, filed Nov. 28, 1989, by Zare et al., on-column conductivity detectors were disclosed wherein on-column sensing electrodes are located contiguous with the exit of the separation microcolumn. The electrodes are covered on their side surfaces with electrical insulators so that conductivity is measured between their ends alone. Another scheme involves covering a crack in the capillary with a porous glass capillary (Wallingford et al., Anal. Chem., 59 (1987), 1762-1766)) to provide off-column amperometric detection.
Although good results have been obtained with current detection systems, they are unsatisfactory in that the structures involved are difficult to fabricate, expensive and, often times, unreliable. This has limited the routine application of both modes of electrochemical detection in CZE. See Ewing et al., Anal. Chem. 61 (1989), 292A-303A; Kuhr, Anal. Chem., 62 (1990), 403R-414R; Huang et al., Anal. Chem., 61 (1989), 766-770; Huang et al., J. Chromatogr., 425 (1988), 385-390; Huang et al., J. Chromatogr., 480 (1989), 285-288; Wallingford et al., Anal. Chem., 60 (1988), 1972-1975; Wallingford et al., Anal. Chem., 60 (1988), 258-263; Wallingford et al., J. Chromatogr., 441 (1988), 299-309; and Wallingford et al., Anal Chem., 61 (1989) 98-100.